In recent years, multiphoton microscopy has been used to gain a better understanding of the pathophysiology of Alzheimer's Disease (AD) in intact live mouse brains. We will use transgenic mouse models that develop senile plaques, a dominant marker of the disease, to investigate the functional and structural consequences of these plaques on neurons in vivo. Neuronal function can be investigated with multiphoton microscopy by monitoring the concentration of calcium within a given cell. Intracellular calcium increases exponentially during neuronal activation and can be detected using calcium-sensitive fluorescent probes. These probes increase their brightness, shift their excitation/emission spectra or engage in Fluorescence Resonance Energy Transfer (FRET) to denote a change in calcium concentration. The ability to observe in real-time the functional effects of senile plaques on neuronal activity will provide a novel marker for testing therapeutics with unprecedented spatial and temporal resolution. To achieve this goal we will develop a gene transfer technique that will allow us to introduce a FRET-based, calcium-sensitive genetic construct directly into the adult mouse brain. The protein encoded by this construct robustly fills soma and neuritic processes. This will permit us to investigate homeostatic alterations in calcium concentration caused by plaque deposition. Dynamic calcium transients can also be monitored using this probe, providing the ability to investigate neuronal activation with spine-level resolution in vivo. We also aim to adapt newly published techniques in bulk loading of functional small-molecule dyes for use in adult, transgenic mice. With a large ensemble of neurons stained with such calcium-sensitive indicators, we will determine the functional consequences of plaques on neighboring cells with single-cell resolution. By combining new imaging modalities, disease neurobiology, and systems-level neuroscience, we hope to provide important and unique insight into the pathogenesis of Alzheimer's Disease. SUMMARY: To date, scientists do not understand how networks of individual cells in a living brain malfunction as Alzheimer's Disease progresses. We will use animal models to determine how neural networks are affected and whether specific therapies can lead to recovery of normal function. [unreadable] [unreadable] [unreadable]